Nitric oxide (NO) in vivo is said to be produced from various parts of the body, such as macrophage, endocapillary cells, and neural regions. Its actions include bacteria-killing action, vascular relaxation action and neural information transmission.
Nitric oxide synthase (NOS) mediates when nitric monoxide is produced. NOS is classified into three groups such as inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) according to the place where it acts. Especially, NO produced by iNOS maintains its activity for long time and the production amount of NO is large (more than 1 μM) as compared with NO produced by the other synthetic system of NO; neuronal NOS and endothelial NOS.
iNOS is mainly expressed when macrophage, endocapillary cell, vessel smooth muscle cell, gastrointestinal epithelia cell, bronchial epithelial cell, hepatocyte, microglial cell or the like is attacked by inflammatory cytokines such as IL-1β (Interleukin-1β), TNF-α (Tumor Necrosis Factor-α) and, IFN-γ (Interferon-γ) which are produced by cytotoxin, infection or inflammatory focus, to produce NO. Excessively produced NO performs as a main inflammatory mediator in the protective response to infection in the live body.
The expression of iNOS occurs along with inflammatory reaction, or the infection of various pathogens such as fungus, virus and protozoa. For example, it is known that iNOS is induced by stimulation of a universal component of gram-negative bacterium, lipopolysaccharide (LPS), or a component of gram-positive bacterium cell walls, lipoteichoic acid (LTA), or through indirect production induction of inflammatory cytokines. Recently, it has been revealed that NO synthesis is increased by in vivo propagation of various viruses. In this case, it has been found that iNOS is induced through production of inflammatory cytokines such as IL-1β or IFN-γ.
NO derived from iNOS shows bacteria-killing, antiviral, antiparasite and antineoplastic activities and is essential to life in biological system. However, if iNOS is activated by inflammatory reactions and the like to produce excessive NO, the NO reacts with active oxygen to generate peroxynitrite or nitrite peroxide which damages DNAs, resulting in negative effects such as mutation and cancerogenesis.
It has been found that excessive expression of iNOS due to various factors which leads to excessive production of NO, causes toxic shock, or various diseases such as systemic decreased blood pressure, decrease in blood pressure response, auto immune diseases, inflammation, arthritis, rheumatic arthritis, diabetes, inflammatory bowel disease, vessel dysfunction, pathogenic vascular dilatation, tissue failure, ischemia of cardiovascular system, hyperpathia, cerebral ischemia, cachexia, and cancer.
Accordingly, controlling the expression of iNOS is very important in biological defense, and in treatment and prevention of diseases related to excessive production of NO, such as cancerogenesis, inflammatory disease, endotoxin shock through bacterial infection or the like.
Conventionally, a method called RNAi (RNA interference), where transcription is repressed by cutting target mRNA in a double-stranded RNA, has been known. Usually, RNAi involves base length of about 20 base pairs and a screening method for its double-stranded oligonucleotide and antisense RNA has been disclosed (Patent Document 1: Japanese Patent Application Laid-Open No. 2005-13224).
Also, compounds useful in controlling interleukin genes, interleukin superfamily genes, expression of genes and/or expression and activities of genes involved in interleukin routes of activities, by RNA interference (RNAi) using small nucleic acid molecules such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules have been disclosed (Patent Document 2:Japanese Patent Application Laid-Open No. 2005-524393).
If antisense RNA exists in a cell, gene expression can be inhibited by hybridizing with complementary mRNA to inhibit translation from mRNA to proteins. If antisense RNA is artificially introduced into a cell, expression of target gene can be inhibited. Therefore, this technique is currently used as a technique shedding light on gene functions and its application into medicinal products has been studied. With respect to iNOS gene, existence of antisense RNA has not been confirmed so far. Further, existence of protein which contributes to stabilization of mRNA in transcription from mRNA has been suggested (non-patent document 1: Eur. J. Pharmacol. 500: 255-266 (2004)). However, the detail is unclear, and there are many points unclear as to controlling of the expression of iNOS.    [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-13224    [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-524393    [Non-patent Document 1] Eur. J. Pharmacol. 500: 255-266 (2004)